Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:137502483-137502610 128bp TCAATTAGCCCCGCCTCT GTACTCTCCGCCTCCTTTCC
Mutant= 130 bp
Wild Type = 128 bp
Wt sequence deletion in lower case
GAGAACTGGGCGATCCACGCCACGCCCCTCTTGGGCCCGCCTCTGTCAATTAGCCCCGCCTCTGCTAGCTAGTCCCGCCCTTCGAAGCGCTCACTAGAACCCTGCgaaccctcaagtctcgcgagccttaaatcgccttgcgcacgcgcacgaggaaaggaggcggagagtacggaagcctagaaggccataagcttatcggttccggcttggagcggggacgcgcgtgcgcagtgcgtagcccttgctggcgagcctgcccggcgttcctcttccgatcccttctccgttcttcttctgcatatcgaggccagcgacgtccacttggatcggtcggcgaggctcacgcgagtgaagggcactgaggaaggaagaaggtaggcgtggacacgccgggaatgggaaccacgtgatgaatgaattaaaactgggagtggagaaagcggaagtggaagggcttgagacactgggaaggaagagggtgttgagggcgcccccttctggttgcacccttagaaagccgggcaagaaaggattctcctgacctccctgaatcttgttccttctccatgccaggactcagagcatggcagaaaacagagaaccccgctgtgccatcgaggctgagctggaccctgtggagtacacccttcggaagcgacttccccaccgcttgccccggaggcccaacgacatttatgtcaacatgaagactgactttaaggcccagctagctcgctgccaaaagctcctggacggaggcactagggggcagaacgcatgcactgaaatctacattcatggcttgggcctggccatcaaccgggccatcaacatcgccctgcagctgcaggcgggcagctttgggtccctacaggtggccgccaatacctccaccgtggagctggtggacgagttggagccagagactgactctcgggagcccctgacgagggtcaggaacaactcagccatccacatccgagtcttcagggtcacgcccaagtaactgaagggaggctgacttggacccaccaaggcttctgagcacacacatccatgtggccgaggtttctagtgcttcatagtggctcttgttctgagttctgtcctgagcctcttgttaccttgttacttcagcctagaggactgagaatggagagcccgagaagtgaatcttccttgtcctgtataataaattattaagttgtgttggtgccgggcttcgaatggcctctttttcctagtgtaattctgtgtgtacttgaataggaaaatggtaggaaagagcaagtatataggccacagtaaggcatctcacattgaaccacagTATGACGGGAATCAAATTTGTTGAGTGTAACACGGAAGCTTAATTGTTACAGTGTGCTCTCAGGAAGATGAGAGAGTAGACTCATCGACTGAGCT
This is a 1228 bp deletion beginning at Chromosome 5 position 137,501,323 bp and ending after 137,502,550 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47452 | TCA ATT AGC CCC GCC TCT | Common | A | |||
47453 | GTA CTC TCC GCC TCC TTT CC | Wild type Reverse | A | |||
47454 | CAT CTT CCT GAG AGC ACA CTG | Mutant Reverse | A | |||
47455 | Fluorophore-1 | AGA ACC CTG CTA TGA CGG G | Quencher-1 | MUT Probe | ||
47456 | Fluorophore-2 | TCA AGT CTC GCG AGC CTT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47452 | 0.40 uM |
47453 | 0.40 uM |
47454 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |